Flap Endonucleases, 5'-3' Exonucleases and 5' Nucleases 

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    A brief  introduction to structure-specific or flap endonucleases (FEN) from the Sayers Laboratory, Sheffield, UK

FEN Biology

FEN Activity

FEN Biotech

FEN Papers

FEN Structures
FEN structure see 5HP4.pdb          Rotating T5FEN 
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RECENT FEN PAPERS FROM SHEFFIELD UNIVERSITY


Conformation change



Threading observed in crystal structures, click here to see the Nature Structural & Molecular Biology paper.
Bending & Binding in substrate recognition see J. Biol. Chem. Human FEN1 bound to DNA see Cell paper from Grasby & Tainer labs E. coli FEN with bound DNA from the Artymiuk Lab see Nucleic Acids Research Mechanism of FEN inhibitors in Nature Chemical Biology

Branched DNA and FEN Biology:

 Not Just a Plot from  "The X Files"

Branched DNA has appeared in "The X Files", supposedly implanted into Agent Dana Scully by aliens so they could track her. Far from being science fiction, branched DNA plays important roles in biology. We usually think about our DNA as being double-helical in structure, but in fact, DNA adopts a number of different shapes as part of normal bilogical processes. Flap endonucleases, 5' nucleases or 5'-3' exonucleases are some of the names given to a group of ubiquitous structure-specific nucleases that can cleave branched DNA, thus restoring the classical double helix. They occur in all living organisms from bacteria to Homo sapiens (see panel above). Some viruses even carry genes encoding their own flap endonuclease enzymes. In the last few years most scientific papers describing these enzymes call them flap endonucleases (FENs), so that is what we will call them here.  Apart from being essential for all cells (they participate in DNA replication and repair processes, e.g. see review by Lewis et al 2016), they are also widely used in biotechnology in genotyping, quantitative PCR, polymorphism screening and molecular biology.

The DNA Pol1 FEN domain was known as the small fragment and was originally described as having 5'-3' exonuclease activity. However, this is indeed a FEN as can be appreciated from the crystal structures FENs and the Thermus aquaticus DNA PolI.FENs are metalloenzymes, with binding sites for 2 or 3 divalent metal ions (see Syson et al). They bind but do not cut DNA in the absence of a suitable divalent metal ion. These enzymes can use a range of divalent metal cofactors ranging incuding  Mg, Mn, Co, Ni, Fe, Ni, Zn and even Cu  (see Garforth & Sayers, Feng et al). The core structure consists of a central beta sheet with a number of helices adorning it. The active site contains several conserved carboxylates (mostly aspartic acid residues), a conserved tyrosine and important lysine and arginine residues.


DNA polymerase I possesses a flap endonuclease domain in addition to the well known Klenow (or large) fragment carrying the polymerase and proofreading polymerase domains. The DNA Pol1 FEN domain was known as the small fragment and was originally described as having 5'-3' exonuclease activity. However, this is indeed a FEN as can be appreciated from the crystal structures FENs and the Thermus aquaticus DNA PolI.FENs are metalloenzymes, with binding sites for 2 or 3 divalent metal ions (see Syson et al). They bind but do not cut DNA in the absence of a suitable divalent metal ion. These enzymes can use a range of divalent metal cofactors ranging incuding  Mg, Mn, Co, Ni, Fe, Ni, Zn and even Cu  (see Garforth & Sayers, Feng et al). The core structure consists of a central beta sheet with a number of helices adorning it. The active site contains several conserved carboxylates (mostly aspartic acid residues), a conserved tyrosine and important lysine and arginine residues.

There are a number of good reviews on biological roles of the FENs (e.g. Bob Bambara's  Ann. Rev. Biochem. or Peter Burgers' JBC review). Basically, at least one FEN is required for cell viability as has been demonstrated in mammalian and bacterial cells. For example FEN knockout mice fail to develop through embryogenesis (Kucherlapati et al) and the FEN domain of PolI is required for cell viability in Streptococcus pneumoniae (Diaz et al). The situation was a little confused regarding bacterial FENs until 2007. For example, Cathy Joyce at Yale showed that the gene encoding DNA PolI (the polA gene) can be deleted in E. coli resulting in bacteria that can grow, albeit slowly on minimal media yet Pol1 was essential for Streptococcus pneumoniae. Joyce also showed that adding back a gene encoding just the FEN-domain of PolI was enough to restore full viability (see Joyce & Grindley, 1984). However, at the time she did not know that many bacteria contain a second FEN-encoding gene (see Allen et al) which I hypothesized might be a backup for the polA-encoded FEN function in 1994 (Sayers, 1994).   Indeed, this seems to be the case and late in 2007, Fukushima et al showed that bacteria require at least one functional FEN activity for viability ( Fukushima et al 2007).

So what do FENs do in the cell? They appear to play major roles in processing the remnants of the RNA primers that are used to initiate Okazaki fragment synthesis (so-called lagging strand synthesis), in maintaining genome stability and in DNA repair (e.g. see Greene et al  and Lindahl & Wood and the reviews above).
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FEN-Catalyzed Reactions

FLAP Endonuclease Activity

The main activity of the FENs is of course their flap endonuclease activity. In its simplest form FENs can cleave 5' flap or "pseudo Y" structures one nucleotide into the double-stranded region immediately downstream of a single-stranded 5' arm. They can also carry out exonucleolyticactivity on free 5' ends of single-stranded or double-stranded DNA. Single strands of DNA are represented as black lines for simplicity, parallel lines indicate double-helical DNA.
FEN ractions

The length of the 5' arm can be anything from one or a few nucleotides to several hundred.  The exonucleotytic products are usually short, 1 to 3 nucleotides in length but there does seem to be some dependence upon the sequence of the single-stranded DNA. For example, oligo G tracts resist cleavage (probably due to quadruplex formation Sayers & Eckstein, JBC).

Gap endonuclease

GAP Endonuclease

Some FENs can cleave single-stranded closed-circular DNA such as the Taq Polymerase-associated FEN and bacteriophage T5 FEN (also known as T5 D15 5'-3' exonuclease see Sayers & Eckstein). They have also been reported to have "gap specific endonuclease" activity or GEN. The term gap endonuclease (GEN) was coined by Shen and coworkers in 2005 when they observed such an activity in human FEN but such and activity was observed for T5FEN much earlier (Sayers & Eckstein 1991).  DNA is represented here as a black line for simplicity, parallel lines represent double-helical DNA.


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FEN Structures

The first FEN structure to be solved was by Tom Steitz's group in 1995. It revealed much of the core FEN structure but not all conserved residues were visible. In 1996, within a week of one another, the structures of the T4 RNase H (a FEN despite its name) was reported by Hosfield et al in Cell and we reported a complete structure of T5 FEN in Nature.
A FEN structure

Images on the left show the structure of the T5FEN molecule and a close-up of the active site. The crystal structure of T5 FEN Ceska, Sayers, Stier and Suck, Nature 1996. PDB code 1EXN. It contains conserved residues that we have mutated in order to ascertain their role in FEN function. For example see our results published in Nature Structural and Molecular Biology, PNAS Dervan, PNAS Garforth.

Since then, several more FEN structures have been reported, but all share the same structural core.  Search the Protein Data Bank for an up to date list using the search term "flap + endonuclease". 


The crystal structure of E. coli Exonuclease IX (the xni gene product) were  solved in  collaborations with Prof. Pete Artymiuk, now we have observed single stranded DNA threaded through another FEN, from bacteriophage T5 (the T5FEN or D15 5'-3' exonuclease).

ExoIX
Enzyme Product Complex. See Anstey-Gilbert CS et al.  It reveals the presence of two very closely spaced Mg ions as well as a potassium ion.  Structures with and without DNA were obtained.  See Almalki et al.  Shows branched DNA threading through the T5FEN. See

 

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FEN Assays and Uses in Biotechnology & Genotyping

The FENs have been used (albeit unwittingly) since the early days of molecular biology. In quantitative RNA assays such as  Third Wave's Invader assay, Applied BiosystemsTaqman  genotyping assays etc are widely used. There is a nice simple animation of the Taqman assay on the Sciencelauncher site. The FEN encoded by bacteriophage T5 can be used to destroy anything apart from pure closed circular plasmid, a useful tool for increasing transfection efficiency and reducing background in cloning experiments. Historically, Amersham's highly efficient site directed mutagenesis (Sculptor) based on the Eckstein labs phosphorothioate method used the T5FEN and has over 60,000 google hits! Sadly, production of the kit has been discontinued. However, contact me if you are interested in resurrecting the phosphorothioate approach.

The popular Gibson cloning system uses T5 exonuclease to allow high-efficiency DNA end-linking as developed by Daniel Gibson at the JCVI in 2009 (Gibson et al. 2009).

We have suggested that the T5FEN is useful for removing linear and nicked (anything not closed circular double-stranded plasmid) from plasmid preps. It can lead to higher transfection rates (Kiss Toth et al) and lower backgrounds in cloning (Sayers et al). Contact me if you need some protein.

In ongoing collaborations led by Jane Grasby, Tim Pickering developed single-turnover substrates for assaying FENs, work which proved the foundation for fluorescent, steady-state and single-turnover assays which use dye-labelled oligonucleotides rather than radio-labelled substrates.

FEN Research in the Sayers Laboratory

Our research has been sponsored by grants from the Wellcome Trust and Biotechnology & Biological Sciences Research Council UK who have funded grants and studentships in this area. We continue to use a combination of site-directed mutagenesis, biochemical and biophysical assays to try to understand the molecular mechanisms operating in these interesting metalloenzymes. We collaborate with kineticist Jane Grasby in the Dept. of Chemistry and with crystallographer Pete Artymiuk in the Dept. of Molecular Biology & Biotechnology, both within the University of Sheffield.

Literature & Links

Search For Papers on FENs by the Sayers' Lab.
Search for papers on all FENs
Link to Jon Sayers' University web pages. Dr Jane Grasby's Pages Kinetics of FENs
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Research in the Sayers' laboratory has funded by;

The Medical Research Council

Meningitis Research Foundation

Biotechnology &  Biological
Sciences Research Council

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Asterion Ltd

The Wellcome Trust


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(C) Jon Sayers, University of Sheffield, all rights reserved. Authored using the Kompozer software on Apple Mac.                                                                         RETURN TO TOP OF PAGE